The overall aim of the proposed research is to develop chemical and enzymatic approaches to study nucleic acid structure and nucleic acid-protein interactions. The present studies focus on the interactions of the restriction and modification enzymes with the specific DNA sequences. To study the mode of action, specificity of several restriction and modification enzymes, DNA's of well defined sequences will be chemically synthesized. The synthetic DNA's will carry selected nucleotide sequences recognized by restriction and modification enzymes. For studies of enzyme-DNA complexes, a DNA pseudo-substrate will be synthesized in which the phosphodiester bond at the site of action will be replaced by methylphosphonate diester linkage. Such a complex will then be crystallized and used for X-ray diffraction analysis. Chemical methods will be developed for the synthesis of short DNA's (8 to 10 nucleotide long duplexes) carrying chemically or photoreactive groups. Such modified substrates will then be used to affinity label the active site of the enzyme. The covalent complex thus obtained will be used: 1) for mapping the active site of the enzyme; and 2) for crystallization to study the three-dimensional structure by X-ray diffraction.